Progressive chromatin silencing of ABA biosynthesis genes permits seed germination in Arabidopsis.

Seed germination represents a major developmental switch in plants that is vital to agriculture, but how this process is controlled at the chromatin level remains obscure. Here we demonstrate that successful germination in Arabidopsis thaliana requires a chromatin mechanism that progressively silences 9-CIS-EPOXYCAROTENOID DIOXYGENASE 6 (NCED6), which encodes a rate-limiting enzyme in abscisic acid (ABA) biosynthesis, through the cooperative action of the RNA-binding protein RZ-1 and the polycomb repressive complex 2 (PRC2). Simultaneous inactivation of RZ-1 and PRC2 blocked germination and synergistically derepressed NCEDs and hundreds of genes. At NCED6, in part by promoting H3 deacetylation and suppressing H3K4me3, RZ-1 facilitates transcriptional silencing and also an H3K27me3 accumulation process that occurs during seed germination and early seedling growth. Genome-wide analysis revealed that RZ-1 is preferentially required for transcriptional silencing of many PRC2 targets early during seed germination, when H3K27me3 is not yet established. We propose RZ-1 confers a novel silencing mechanism to compensate for and synergize with PRC2. Our work highlights the progressive chromatin silencing of ABA biosynthesis genes via the RNA-binding protein RZ-1 and PRC2 acting in synergy, a process that is vital for seed germination.

Transcriptional Gene Silencing Maintained by OTS1 SUMO Protease Requires a DNA-Dependent Polymerase V-Dependent Pathway.

The expression of genes with aberrant structure is prevented at both the transcriptional and posttranscriptional regulation levels. Aberrant gene silencing at the posttranscriptional level is well studied; however, it is not well understood how aberrant genes are silenced at the transcriptional level. In this study, through genetic screening a transgenic report line that harbors an aberrant gene (35S-LUC, lacking 3'-untranslated region [3'-UTR]) and lacks luciferase (LUC) activity, we identify that the small ubiquitin-like modifier (SUMO) protease OTS1 gene is required for maintaining the silence of the reporter 35S-LUC and an endogenous mutator-like element MULE-F19G14 at the transcriptional level, which requires DNA-dependent RNA polymerase (Pol) V and DDR complex, but not Pol IV. The increased transcripts in ots1 mutants are terminated by the 3'-UTRs of downstream genes. In addition to ots1 mutations, mutations in several known or putative SUMO proteases and two SUMO E3 ligases, SIZ1 and MMS21, have similar effects on this silencing regulation. Taken together, our results reveal that the enzymes involved in the SUMOylation process restrain aberrant gene transcription by using a downstream gene 3'-UTR, and this regulation requires a functional Pol V-dependent pathway in Arabidopsis (Arabidopsis thaliana).

Image Registration-Based Bolt Loosening Detection of Steel Joints.

Self-loosening of bolts caused by repetitive loads and vibrations is one of the common defects that can weaken the structural integrity of bolted steel joints in civil structures. Many existing approaches for detecting loosening bolts are based on physical sensors and, hence, require extensive sensor deployment, which limit their abilities to cost-effectively detect loosened bolts in a large number of steel joints. Recently, computer vision-based structural health monitoring (SHM) technologies have demonstrated great potential for damage detection due to the benefits of being low cost, easy to deploy, and contactless. In this study, we propose a vision-based non-contact bolt loosening detection method that uses a consumer-grade digital camera. Two images of the monitored steel joint are first collected during different inspection periods and then aligned through two image registration processes. If the bolt experiences rotation between inspections, it will introduce differential features in the registration errors, serving as a good indicator for bolt loosening detection. The performance and robustness of this approach have been validated through a series of experimental investigations using three laboratory setups including a gusset plate on a cross frame, a column flange, and a girder web. The bolt loosening detection results are presented for easy interpretation such that informed decisions can be made about the detected loosened bolts.

Arabidopsis small ubiquitin-related modifier protease ASP1 positively regulates abscisic acid signaling during early seedling development.

The small ubiquitin-related modifier (SUMO) modification plays an important role in the regulation of abscisic acid (ABA) signaling, but the function of the SUMO protease, in ABA signaling, remains largely unknown. Here, we show that the SUMO protease, ASP1 positively regulates ABA signaling. Mutations in ASP1 resulted in an ABA-insensitive phenotype, during early seedling development. Wild-type ASP1 successfully rescued, whereas an ASP1 mutant (C577S), defective in SUMO protease activity, failed to rescue, the ABA-insensitive phenotype of asp1-1. Expression of ABI5 and MYB30 target genes was attenuated in asp1-1 and our genetic analyses revealed that ASP1 may function upstream of ABI5 and MYB30. Interestingly, ASP1 accumulated upon ABA treatment, and ABA-induced accumulation of ABI5 (a positive regulator of ABA signaling) was abolished, whereas ABA-induced accumulation of MYB30 (a negative regulator of ABA signaling) was increased in asp1-1. These findings support the hypothesis that increased levels of ASP1, upon ABA treatment, tilt the balance between ABI5 and MYB30 towards ABI5-mediated ABA signaling.

Arabidopsis SUMO protease ASP1 positively regulates flowering time partially through regulating FLC stability .

The initiation of flowering is tightly regulated by the endogenous and environment signals, which is crucial for the reproductive success of flowering plants. It is well known that autonomous and vernalization pathways repress transcription of FLOWERING LOCUS C (FLC), a focal floral repressor, but how its protein stability is regulated remains largely unknown. Here, we found that mutations in a novel Arabidopsis SUMO protease 1 (ASP1) resulted in a strong late-flowering phenotype under long-days, but to a lesser extent under short-days. ASP1 localizes in the nucleus and exhibited a SUMO protease activity in vitro and in vivo. The conserved Cys-577 in ASP1 is critical for its enzymatic activity, as well as its physiological function in the regulation of flowering time. Genetic and gene expression analyses demonstrated that ASP1 promotes transcription of positive regulators of flowering, such as FT, SOC1 and FD, and may function in both CO-dependent photoperiod pathway and FLC-dependent pathways. Although the transcription level of FLC was not affected in the loss-of-function asp1 mutant, the protein stability of FLC was increased in the asp1 mutant. Taken together, this study identified a novel bona fide SUMO protease, ASP1, which positively regulates transition to flowering at least partly by repressing FLC protein stability.

SUMO E3 Ligases GmSIZ1a and GmSIZ1b regulate vegetative growth in soybean .

SIZ1 is a small ubiquitin-related modifier (SUMO) E3 ligase that mediates post-translational SUMO modification of target proteins and thereby regulates developmental processes and hormonal and environmental stress responses in Arabidopsis. However, the role of SUMO E3 ligases in crop plants is largely unknown. Here, we identified and characterized two Glycine max (soybean) SUMO E3 ligases, GmSIZ1a and GmSIZ1b. Expression of GmSIZ1a and GmSIZ1b was induced in response to salicylic acid (SA), heat, and dehydration treatment, but not in response to cold, abscisic acid (ABA), and NaCl treatment. Although GmSIZ1a was expressed at higher levels than GmSIZ1b, both genes encoded proteins with SUMO E3 ligase activity in vivo. Heterologous expression of GmSIZ1a or GmSIZ1b rescued the mutant phenotype of Arabidopsis siz1-2, including dwarfism, constitutively activated expression of pathogen-related genes, and ABA-sensitive seed germination. Simultaneous downregulation of GmSIZ1a and GmSIZ1b (GmSIZ1a/b) using RNA interference (RNAi)-mediated gene silencing decreased heat shock-induced SUMO conjugation in soybean. Moreover, GmSIZ1RNAi plants exhibited reduced plant height and leaf size. However, unlike Arabidopsis siz1-2 mutant plants, flowering time and SA levels were not significantly altered in GmSIZ1RNAi plants. Taken together, our results indicate that GmSIZ1a and GmSIZ1b mediate SUMO modification and positively regulate vegetative growth in soybean.

Reversible SUMOylation of FHY1 Regulates Phytochrome A Signaling in Arabidopsis.

In response to far-red light (FR), FAR-RED ELONGATED HYPOCOTYL 1 (FHY1) transports the photoactivated phytochrome A (phyA), the primary FR photoreceptor, into the nucleus, where it initiates FR signaling in plants. Light promotes the 26S proteasome-mediated degradation of FHY1, which desensitizes FR signaling, but the underlying regulatory mechanism remains largely unknown. Here, we show that reversible SUMOylation of FHY1 tightly regulates this process. Lysine K32 (K32) and K103 are major SUMOylation sites of FHY1. We found that FR exposure promotes the SUMOylation of FHY1, which accelerates its degradation. Furthermore, we discovered that ARABIDOPSIS SUMO PROTEASE 1 (ASP1) interacts with FHY1 in the nucleus under FR and facilitates its deSUMOylation. FHY1 was strongly SUMOylated and its protein level was decreased in the asp1-1 loss-of-function mutant compared with that in the wild type under FR. Consistently, asp1-1 seedlings exhibited a decreased sensitivity to FR, suggesting that ASP1 plays an important role in the maintenance of proper FHY1 levels under FR. Genetic analysis further revealed that ASP1 regulates FR signaling through an FHY1- and phyA-dependent pathway. Interestingly, We found that continuous FR inhibits ASP1 accumulation, perhaps contributing to the desensitization of FR signaling. Taken together, these results indicate that FR-induced SUMOylation and ASP1-dependent deSUMOylation of FHY1 represent a key regulatory mechanism that fine-tunes FR signaling.

SIZ1-Mediated SUMOylation of TPR1 Suppresses Plant Immunity in Arabidopsis.

Plant immune responses are tightly regulated to ensure their appropriate deployment. Overexpression of TOPLESS-RELATED 1 (TPR1), a SUPPRESSOR OF npr1-1, CONSTITUTIVE 1 (SNC1)-interacting protein, results in autoimmunity that reduces plant growth and development. However, how TPR1 activity is regulated remains unknown. Loss of function of SIZ1, a (SUMO) E3 ligase, induces an autoimmune response, partially due to elevated SNC1 levels. Here we show that SNC1 expression is upregulated in Arabidopsis thaliana siz1-2 due to positive-feedback regulation by salicylic acid. SIZ1 physically interacts with TPR1 and facilitates its SUMO modification. The K282 and K721 residues in TPR1 serve as critical SUMO attachment sites. Simultaneous introduction of K282R and K721R substitutions in TPR1 blocked its SUMOylation, enhanced its transcriptional co-repressor activity, and increased its association with HISTONE DEACETYLASE 19 (HDA19), suggesting that SUMOylation of TPR1 represses its transcriptional co-repressor activity and inhibits its interaction with HDA19. In agreement with this finding, the simultaneous introduction of K282R and K721R substitutions enhanced TPR1-mediated immunity, and the tpr1 mutation partially suppressed autoimmunity in siz1-2. These results demonstrate that SIZ1-mediated SUMOylation of TPR1 represses plant immunity, which at least partly contributes to the suppression of autoimmunity under non-pathogenic conditions to ensure proper plant development.